With the establishment of the general principle of lipid assisted folding in the organization of several polytopic membrane proteins, the proposed studies are aimed at defining in molecular detail the determinants within the membrane protein structure and the lipid environment that govern directional organization of alpha-helical transmembrane-spanning domains (TMs). When expressed in cells lacking phosphatidylethanolamine (PE), the N-terminal six TMs of LacY (lactose permease) and N-terminal two TMs of PheP (phenylalanine permease) are mis-oriented with respect to the membrane bilayer. Reversal of mis-orientation for both occurs in vivo upon post-assembly synthesis of PE. Since the topological organization of these two proteins respond to the lipid environment in different ways, the determinants that affect their organization will be compared and contrasted to provide a broader coverage of possible mechanisms. In Specific Aim 1 the organization of LacY in PE-lacking cells will be more fully defined as a basis for investigating the determinants of topological organization. A more detailed characterization of the reversibility of LacY topological organization as a function of membrane lipid composition will be carried out. In Specific Aim 2 both a genetic and site-directed mutagenesis approach will be used to identify the signals within the sequences of LacY and PheP that interact in concert with the lipid environment to determine topology of TMs. In Specific Aim 3 the effects different membrane lipid compositions, both changes in native lipids and introduction of non-native lipids, on topological organization of TMs of these two proteins will be determined in order to elucidate the properties of lipids that affect TM orientation. In Specific Aim 4 the native proteins and mutants isolated in Specific Aim 2 will be reconstituted into proteoliposomes of defined lipid composition based on results from Specific Aim 3 in order to study the direct effects of protein signals and lipid environment in a controlled system without interference by other cellular components. The reversibility of TM orientation of LacY and PheP as a function of changes in lipid composition will also be studied to determine if other cellular components are required. The results of these studies will provide a set of rules that govern how lipid-protein interactions determine TM organization of membrane proteins. Understanding the rules for protein folding has important implications for understanding a number of diseases involving mis-folded proteins such as cystic fibrosis, Alzheimer's disease, and other dementias.